Zheng Libin, Huang Dong
Guangdong Medical University, Zhanjiang Guangdong, 524023, P.R.China.
Guangdong Medical University, Zhanjiang Guangdong, 524023, P.R.China;Department of Trauma Orthopedics, Guangdong Second Provincial Central Hospital, Guangzhou Guangdong, 510000,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2018 May 15;32(5):575-580. doi: 10.7507/1002-1892.201710109.
To investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts.
Murine RAW264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D , and identified by tartrate resistant acid phosphatase (TRAP) staining. Then, the osteoclasts were divided into 2 groups. The osteoclasts were treated with 400 ng/mL FTY720-P in experimental group and without FTY720-P in control group, respectively. After 48 hours of culture, the cells in 2 groups were detected by real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining. The expressions of EphA2, EphrinA2, RhoA, and the bone reconstruction associated proteins［bone morphogenetic protein 2 (BMP-2) and transform growth factor β (TGF-β )］were analyzed and compared.
RAW264.7 cells were successfully induced into osteoclasts identified by TRAP staining. Compared with control group, the relative expressions of EphA2 and EphrinA2 mRNAs and proteins in experimental group significantly decreased after 48 hours ( <0.05), and the relative expression of RhoA protein also significantly decreased ( <0.05). The relative expressions of BMP-2 and TGF-β mRNAs were significantly increased ( <0.05), and those protein expressions were enhanced.
FTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β and BMP-2 by affecting the transduction of EphA2-EphrinA2 bidirectional signaling in osteoclasts.
研究FTY720-P对破骨细胞中EphA2-EphrinA2双向信号传导的影响。
用地塞米松和1α,25-二羟基维生素D将小鼠RAW264.7巨噬细胞诱导为破骨细胞，并用抗酒石酸酸性磷酸酶（TRAP）染色进行鉴定。然后，将破骨细胞分为2组。实验组用400 ng/mL FTY720-P处理，对照组不使用FTY720-P。培养48小时后，通过实时荧光定量PCR、蛋白质免疫印迹法和免疫荧光染色检测两组细胞。分析并比较EphA2、EphrinA2、RhoA以及骨重建相关蛋白［骨形态发生蛋白2（BMP-2）和转化生长因子β （TGF-β ）］的表达。
经TRAP染色鉴定，RAW264.7细胞成功诱导为破骨细胞。与对照组相比，实验组在48小时后EphA2和EphrinA2 mRNA及蛋白的相对表达显著降低（<0.05），RhoA蛋白的相对表达也显著降低（<0.05）。BMP-2和TGF-β mRNA的相对表达显著增加（<0.05），其蛋白表达增强。
FTY720-P可通过影响破骨细胞中EphA2-EphrinA2双向信号转导，下调RhoA表达，促进TGF-β 和BMP-2的表达。
